Normal human peripheral blood mononuclear cells were immunized in vitro to the soluble antigen tetanus toxoid. Antibody production of IgG and IgM was monitored by ELISA assay. Time, antigen dosage and various culture conditions were evaluated to optimize the antibody response. Cells immunized in vitro were used to make human-human hybridoma fusions and human-mouse hybridoma fusions. Fusions of a human plasma cell tumor with B cells from cultures immunized for 6 to 11 days in vitro resulted in the production of antigen-specific human monoclonal antibodies, mostly of the IgG class. The peak time for fusion slightly preceded the time of maximal antibody production (11 days). Hybrids were stable and continued to produce for at least 60 days. No antibody to a non-crossreacting antigen, keyhole limpet hemocyanin, was detected. Human-mouse hybrids which produced specific human immunoglobulin against tetanus toxoid were also obtained using in vitro immunized human cells as fusion partners. Addition of semi-purified lymphokine preparations could enhance the numbers of antibody-producing cells during sensitization in both the human and murine systems.